JP5321305B2 - Scanning microscope - Google Patents

Description

  The present invention relates to a scanning microscope, and more particularly to a scanning microscope that detects light from a sample without descanning.

  In recent years, in the field of biological microscopes, microscopes using nonlinear effects have attracted attention. In particular, a scanning multiphoton microscope using multiphoton excitation (see, for example, Patent Document 1) is resistant to diffusion and can observe a deep part of a sample, so that the demand from users is increasing. If a scanning multiphoton microscope is used, for example, it is possible to observe a highly diffuse sample such as a brain, which has been difficult to observe.

  Further, in a conventional scanning confocal microscope, laser light (hereinafter referred to as excitation light) is irradiated while scanning, and the observation light emitted from the sample is descanned while passing through a pinhole to achieve a confocal effect. Thus, the resolution in the optical axis direction is obtained. On the other hand, in the scanning multiphoton microscope, only the portion of the sample where the excitation light is condensed is excited, so that resolution in the optical axis direction can be obtained without using a pinhole.

  As described above, in the scanning multiphoton microscope, since it is not necessary to use a pinhole, it is not always necessary to descan the observation light from the sample. Therefore, for example, a light beam separating unit is provided between the scanning unit that scans the excitation light and the objective lens, and the observation light is reflected by the light beam separating unit in a direction different from the excitation light, and the reflected observation light is not descanned. In addition, it is possible to detect by a detecting means such as PMT (photo multiplier tube). An optical system that detects observation light without descanning in a scanning microscope is also referred to as NDD (Non Descanned Detector).

  The conventional scanning confocal microscope can only receive the observation light from the part conjugate with the pinhole of the sample, while the scanning multiphoton microscope using NDD also has scattered light around the excitation light spot. Light can be received and a brighter image can be obtained.

JP 2000-330029 A

  By the way, in the conventional optical system for descanning the observation light, since the incident angle of the light beam incident on the detection means becomes almost 0 degrees, the cover can be simply provided by arranging the barrier filter perpendicular to the light beam incident on the detection means. It was possible to sufficiently prevent the excitation light reflected by glass or the like from entering the detection means.

  On the other hand, in the NDD, since the observation light is detected without descanning, the incident angle of the light beam incident on the detection means is not necessarily 0 degree, and light beams with various incident angles are incident. Therefore, since the incident angle of the light beam entering the barrier filter is not constant, the arrangement of the barrier filter as in the case of the optical system for descanning the observation light sufficiently prevents the excitation light from entering the detection means. It may not be possible.

  The present invention has been made in view of such a situation, and makes it possible to easily and reliably prevent excitation light from entering a detection means for detecting observation light from a sample.

A scanning microscope according to one aspect of the present invention irradiates a sample while scanning excitation light that can be excited by multiphotons through a scanning unit, and does not scan the observation light emitted from the sample by the scanning unit. A scanning microscope for detecting by the detection means , having a characteristic of shielding light in the same wavelength range as the excitation light on the optical path of the observation light to the detection means for detecting the observation light , and A first light shielding means that does not have a property of selectively transmitting observation light; a property of shielding light in the same wavelength region as the excitation light; and a property of selectively transmitting the observation light. An incident angle to the second light shielding means of at least a part of the excitation light that passes through the first light shielding means and is incident on the second light shielding means. It is configured to be different from the incident angle to one light shielding means.

  In the scanning microscope according to one aspect of the present invention, light having the same wavelength region as that of the excitation light is shielded by the first light shielding unit and the second light shielding unit out of the light toward the detection unit that detects the observation light. .

  According to one aspect of the present invention, it is possible to easily and reliably prevent excitation light from entering a detection unit that detects observation light from a sample.

It is a block diagram which shows one Embodiment of the scanning microscope to which this invention is applied. It is a figure which shows the specific example of the optical path of the observation light in the case of using a scanning microscope as a scanning multiphoton microscope. It is a figure which shows an example of the performance of IR cut filter. It is the figure which showed in a straight line the optical path from the objective lens to the focal plane of the lens arrange | positioned between IR cut filters. It is a figure for demonstrating the installation conditions of the lens arrange | positioned between IR cut filters. It is a figure for demonstrating the installation conditions of the lens arrange | positioned between IR cut filters. It is a figure for demonstrating the installation conditions of the lens arrange | positioned between IR cut filters. It is a figure which shows the experimental result which changed the installation conditions of IR cut filter, and compared the light-shielding performance. It is a figure which shows the other example of installation of IR cut filter.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of a scanning microscope to which the present invention is applied.

  The laser light source 11 of the scanning microscope 1 can emit a plurality of laser beams having different wavelengths simultaneously or individually. In the following description, it is assumed that two types of laser light, infrared light and visible light, are emitted from the laser light source 11. In addition, hereinafter, the infrared light emitted from the laser light source 11 is a very short (for example, 100 femtosecond) pulsed light (for example, 100 femtoseconds) emitted in a predetermined cycle for inducing multiphoton excitation of the sample 2. Hereinafter, it is referred to as IR pulse light).

  Laser light emitted from the laser light source 11 passes through the scanning device 12, the condenser lens 13, the dichroic mirror 14, and the objective lens 15, and is applied to the sample 2 installed on the stage 16.

  More specifically, the laser light emitted from the laser light source 11 enters the scanning device 12, passes through the dichroic mirror 41, and enters the scanning unit 42. The scanning unit 42 is configured by, for example, a galvano scanner, and the laser light is deflected by the scanning unit 42, passes through the pupil projection lens 43, and is emitted from the scanning device 12.

  The laser light that has passed through the pupil projection lens 43 is once condensed, then converted into a parallel light beam by the condenser lens 13, transmitted through the dichroic mirror 14, and condensed on the observation surface of the sample 2 by the objective lens 15. At this time, the scanning unit 42 scans the laser beam on the observation surface of the sample 2 while controlling the scanning range and scanning speed of the laser beam based on the control of a control unit (not shown).

  When observing the sample 2, either IR pulse light or visible light is used as excitation light for imaging (excitation) for obtaining an image of the sample 2.

  For example, when IR pulse light is used as excitation light, visible light is not used at all or is used for light stimulation of the sample 2. In this case, when the sample 2 is irradiated with IR pulse light, fluorescence due to multiphoton excitation appears from the sample 2, and this fluorescence becomes observation light, passes through the objective lens 15, and is parallelized by the objective lens 15. The light beam is incident on the dichroic mirror 14, reflected in the direction of an NDD (Non Descaned Detector) 17 by the dichroic mirror 14, and incident on the NDD 17.

  The IR cut filter 61a and the IR cut filter 61b provided in the NDD 17 have the same performance, shield light in a predetermined wavelength region including the same wavelength region as the IR pulse light, and transmit light of other wavelengths. To do. The observation light incident on the NDD 17 is shielded from components in a predetermined wavelength range by the IR cut filter 61a, and other components are transmitted. The observation light that has passed through the IR cut filter 61a is incident on the IR cut filter 61b while being collected by the lens 62. The IR cut filter 61b shields components in a predetermined wavelength region, and transmits other components. .

  The observation light transmitted through the IR cut filter 61b is incident on the dichroic mirror 63, and components other than the predetermined first wavelength band are transmitted through the dichroic mirror 63, and components in the first wavelength band are transmitted by the dichroic mirror 63. Reflected in the direction of the barrier filter 64b. The observation light that has passed through the dichroic mirror 63 is transmitted through the barrier filter 64a by a component in a predetermined second wavelength region, is converted into a parallel light flux by the lens 65a, and enters a PMT (photo multiplier tube) 66a. . The observation light reflected in the direction of the barrier filter 64b is transmitted through the first wavelength band component by the barrier filter 64b, is converted into a parallel light beam by the lens 65b, and enters the PMT 66b.

  Each of the PMTs 66a and 66b detects the received observation light and supplies a voltage detection signal corresponding to the amount of light to a controller (not shown). In addition, since the IR pulse light irradiated to the sample 2 is scanned by the scanning unit 42 as described above, the PMTs 66a and 66b detect observation light over the observation surface of the sample 2. A controller (not shown) generates an observation image of the sample 2 based on each electrical signal from the PMTs 66a and 66b.

  On the other hand, when visible light is used as excitation light, IR pulse light is used for light stimulation of the sample 2 by multiphoton excitation. That is, when the observation surface of the sample 2 is stimulated by the IR pulse light and the observation surface is irradiated with visible light, the sample 2 emits fluorescence, and this fluorescence becomes observation light, and the objective lens. 15, converted into a parallel light beam by the objective lens 15, transmitted through the dichroic mirror 14, once condensed by the condensing lens 13, converted into a parallel light beam by the pupil projection lens 43, and incident on the scanning unit 42. The observation light is descanned by the scanning unit 42 and reflected by the dichroic mirror 41 in the direction of the condenser lens 44. The observation light reflected in the direction of the condenser lens 44 enters the PMT 46 through the pinhole 45 provided at a position substantially conjugate with the focal position of the objective lens 15.

  The PMT 46 detects the received observation light and supplies a detection signal having a voltage corresponding to the amount of light to a controller (not shown). Note that the visible light irradiated to the sample 2 is scanned by the scanning unit 42 as described above, and thus the PMT 46 detects the observation light over the observation surface of the sample 2. A controller (not shown) generates an observation image of the sample 2 based on the electrical signal from the PMT 46.

  Thus, the scanning microscope 1 can be used as both a scanning multiphoton microscope and a scanning confocal microscope.

  Hereinafter, the IR cut filter 61a and the IR cut filter 61b are simply referred to as the IR cut filter 61 when it is not necessary to distinguish between them.

  FIG. 2 is a diagram illustrating a specific example of the optical path of the observation light when the scanning microscope 1 is used as a scanning multiphoton microscope.

  The observation light L1 expressed from the point A1 on the observation surface S of the sample 2 near the optical axis of the objective lens 15 is converted into a parallel light beam by the objective lens 15, passes through the pupil P of the objective lens 15, and passes through the dichroic mirror 14. Is reflected in the direction of NDD17. The observation light L1 incident on the NDD 17 enters the IR cut filter 61a from a substantially vertical direction, passes through the IR cut filter 61a, and then passes through the IR cut filter 61b while being condensed by the lens 62. The light is collected at the focal plane F. The observation light L1 is branched in two directions by the dichroic mirror 63, and the observation light transmitted through the dichroic mirror 63 is transmitted through the barrier filter 64a, converted into a parallel light beam by the lens 65a, and incident on the PMT 66a from a substantially vertical direction. To do. On the other hand, the observation light L1 reflected by the dichroic mirror 63 passes through the barrier filter 64b, becomes a parallel light beam by the lens 65b, and enters the PMT 66b from a substantially vertical direction.

  In addition, the observation light L2 expressed from the point A2 on the observation surface S of the sample 2 at a position shifted from the optical axis of the objective lens 15 enters the NDD 17 in the same manner as the observation light L1. The observation light L2 incident on the NDD 17 enters the IR cut filter 61a from an oblique direction, passes through the IR cut filter 61a, and then passes through the IR cut filter 61b while being condensed by the lens 62. Condensate at F. The observation light L2 is branched in two directions by the dichroic mirror 63, and the observation light transmitted through the dichroic mirror 63 is transmitted through the barrier filter 64a, is converted into a parallel light beam by the lens 65a, and enters the PMT 66a from an oblique direction. On the other hand, the observation light L2 reflected by the dichroic mirror 63 passes through the barrier filter 64b, becomes a parallel light beam by the lens 65b, and enters the PMT 66b from an oblique direction.

  Thus, when the scanning microscope 1 is used as a scanning multiphoton microscope, since the observation light is not descanned, each part of the NDD 17 (the IR cut filter 61 a, depending on the position where the observation light appears on the observation surface S of the sample 2). The incident angles to the IR cut filter 61b, PMT 66a, PMT 66b, etc.) are different.

  Further, part of the IR pulse light used for multiphoton excitation is incident on the NDD 17 as noise light. For example, a part of the IR pulse light that has passed through the pupil P of the objective lens 15 is reflected by the interface of the cover glass (not shown) placed on the sample 2 or the objective lens 15 and has almost the same optical path as the observation light. And enter the NDD 17. Most of the IR pulse light incident on the NDD 17 follows this optical path.

  For example, a part of the IR pulse light does not enter the pupil of the objective lens 15 but is reflected in the lens barrel of the scanning microscope 1 and enters the NDD 17. Most of the light passes through the pupil P of the objective lens 15 and the vicinity thereof and enters the NDD 17.

  Therefore, it can be considered that the IR pulse light (noise light) incident on the NDD 17 passes through the pupil P of the objective lens 15 and enters the NDD 17. The incident angle of the noise light to the NDD 17 also has various values as in the observation light.

  The IR pulse light is a powerful laser light having an optical output of several W, and the amount of noise light is never weak compared to the amount of fluorescence expressed from the sample 2. Therefore, the scanning microscope 1 is provided with two filters, an IR cut filter 61a and an IR cut filter 61b, in order to reliably prevent noise light from entering the PMTs 66a and 66b.

  By the way, in general, the performance of an optical filter varies depending on the incident angle of light. FIG. 3 shows an example of the performance of the IR cut filter 61. In the figure, the horizontal axis represents wavelength, and the vertical axis represents OD (Optical Density, optical density). In addition, the solid line in the figure indicates the light blocking characteristic when the incident angle of the light beam to the IR cut filter 61 is 0 degrees, and the dotted line indicates the light blocking characteristic when the incident angle is 45 degrees.

  As shown in FIG. 3, the light shielding characteristics of the IR cut filter 61, that is, the wavelength range of the light to be shielded and the OD value of the transmitted light are greatly different depending on the incident angle. Becomes larger and the light shielding rate is improved. In the example of FIG. 3, the tendency is particularly prominent in a long wavelength region of 850 mn or more.

  As described above, the incident angle of the noise light to the IR cut filter 61a is various. For example, as with the observation light L1 in FIG. The cut filter 61a can be sufficiently shielded. On the other hand, as with the observation light L2 in FIG. 2, noise light incident obliquely on the IR cut filter 61a may not be sufficiently shielded by the IR cut filter 61a depending on the incident angle. Therefore, in the scanning microscope 1, noise light is removed using not only the IR cut filter 61a but also the IR cut filter 61b.

  Also, most of the noise light transmitted through the IR cut filter 61a is refracted by the lens 62 disposed between the IR cut filter 61a and the IR cut filter 61b, and the incident angle to the IR cut filter 61a and the IR cut filter are reduced. The incident angle to 61b is different. Therefore, compared with the case where the IR cut filter 61a and the IR cut filter 61b are arranged so that the light shielding surfaces are parallel to each other without sandwiching the lens 62, the noise light transmitted through the IR cut filter 61a is more reduced. It can be reliably shielded by the IR cut filter 61b.

  Here, the conditions of the installation position of the lens 62 will be considered with reference to FIGS. FIG. 4 is a diagram showing the optical paths from the objective lens 15 to the focal plane F of the lens 62 arranged in a straight line for easy understanding. In the figure, a light beam L11 indicates a light beam traveling on the same optical path as the observation light L1 in FIG. 2, and a light beam L12 indicates a light beam traveling on the same optical path as the observation light L2 in FIG. Further, hereinafter, the distance between the pupil P of the objective lens 15 and the lens 62 is d, and the focal length of the lens 62 (the distance between the lens 62 and the focal plane F in the figure) is f.

  As described above, for example, when noise light travels on the same optical path as the light beam L11 and enters the IR cut filter 61a from a substantially vertical direction, the noise light is sufficiently shielded by the IR cut filter 61a. On the other hand, when the noise light travels on the same optical path as the light beam L12 and enters the IR cut filter 61a from an oblique direction, the ratio of the noise light transmitted without being blocked by the IR cut filter 61a is increased, and a sufficient light shielding rate is secured. It may not be possible. Therefore, the case where noise light travels on the same optical path as the light beam L12 will be considered below.

  First, when the distance d = focal length f, the principal ray Lm of the light beam L12 is transmitted through the lens 62, becomes substantially parallel to the optical axis of the lens 62, and enters the IR cut filter 61b from a substantially vertical direction. To do. Therefore, in this case, the ratio at which the noise light transmitted through the IR cut filter 61a is shielded by the IR cut filter 61b is maximized.

  Next, consider a case where the focal length f <the distance d.

First, as shown in FIG. 5, the incident angle of the light ray L21 passing through the position of the height h from the center of the lens 62 to the IR cut filter 61a (= the exit angle from the objective lens 15 = the incident to the lens 62). corners) and theta _a, the incident angle of the IR cut filter 61b of the light beam L21 (the exit angle from = lens 62) and theta _b, the relationship between the incident angle theta _b the incident angle theta _a, the following equation It is represented by (1).

Hereinafter, regarding the angles in the figure, the clockwise direction is defined as a positive direction, and the counterclockwise direction is defined as a negative direction. Accordingly, in FIG. 5, the incident angle θ _a is a negative value, and the incident angle θ _b is a positive value.

Then, when the following equation (2) holds, i.e., when the magnitude of the incident angle theta _b (absolute value) is smaller than the size of the incident angle theta _a (absolute value), the process proceeds along the same optical path as the light beam L21, IR The noise light transmitted through the cut filter 61a is considered to be sufficiently shielded by the IR cut filter 61b.

As described above, it can be considered that the noise light incident on the NDD 17 substantially passes through the pupil P of the objective lens 15. Therefore, when the focal length f is shorter than the distance d, the magnitude of the incident angle theta _a to IR cut filter 61a of the noise light is maximized, for example, as the light L31 in FIG. 6, the objective lens 15 This is when passing through the lower end of the pupil P and passing through the upper end of the effective diameter of the lens 62. Then, when the magnitude of the incident angle theta _a is maximum, if so Equation (2) holds, regardless of the magnitude of the incident angle theta _a, a noise light that has passed through the IR cut filter 61a, an IR cut It is considered that the filter 61b can be sufficiently shielded.

Here, the radius of the pupil P of the objective lens 15 recommended for observation is h ₁ , the radius of the effective diameter of the lens 62 is h ₂ , the incident angle θ _a is small, and θ _a ≈tan θ _a = − (h ₁ + h ₂ ) / d, the equation (2) can be transformed into the following equation (3) for the light ray L31 in FIG.

  Then, when formula (3) is arranged so that the focal length f is on the left side, the following formula (4) is obtained.

  Therefore, when the focal distance f is shorter than the distance d, when the relationship between the distance d and the focal distance f satisfies Expression (4), the noise light transmitted through the IR cut filter 61a is sufficiently shielded by the IR cut filter 61b. It is considered possible.

  Next, consider the case where the focal length f> the distance d. In this case, if the light transmitted through the lens 62 forms an image with a size larger than the effective diameter of the lens 62 on the focal plane F of the lens 62, the size of the NDD 17 needs to be increased.

For example, as shown in FIG. 7, the imaging position on the focal plane F of the light beam L41 passing through the upper end of the effective diameter of the lens 62 is x, and the distance between the imaging position x and the optical axis of the lens 62 is defined as x. If h ₃ , the distance H ₃ > the effective diameter radius h ₂ of the lens 62 needs to increase the size of the NDD 17. This incident angle theta _b to IR cut filter 61b of the light beam Lu of the upper end of the light beam L41 is a case where less than 0. Therefore, when the focal length f is longer than the distance d, the incident angle θ _b of the light beam Lu to the IR cut filter 61b may satisfy the following formula (5). That is, the light beam Lu may be transmitted in the horizontal direction or obliquely downward in the figure after passing through the lens 62.

In the equation (5), θ _a represents an incident angle of the light beam Lu to the IR cut filter 61a. Here, if the incident angle θ _a is small and θ _a ≈tan θ _a = − (h ₂ −h ₁ ) / d holds, the equation (5) can be transformed into the following equation (6).

  Then, when formula (6) is arranged so that the focal length f is on the left side, the following formula (7) is obtained.

  Combining the above equations (4) and (7) yields the following equation (8).

  Here, when the pupil diameter φ1 of the objective lens 15 and the effective diameter φ2 of the lens 62 are used instead of the radius h1 of the pupil P of the objective lens 15 and the radius h2 of the effective diameter of the lens 62, Expression (8) is Equation (9) is obtained.

  It is assumed that the pupil diameter φ1 of the objective lens 15 <the effective diameter φ2 of the lens 62 (the radius h1 of the pupil P of the objective lens 15 <the radius h2 of the effective diameter of the lens 62).

  Therefore, when the relationship between the distance d and the focal length f satisfies the equation (9), it is possible to reliably prevent noise light from entering the PMT 66a and the PMT 66b without increasing the size of the NDD 17.

  In addition, when the formula (9) is arranged with respect to the distance d, the following formula (10) is obtained.

  The installation conditions for the lens 62 described above are just an example. For example, the installation conditions may be obtained in consideration of other parameters such as the performance of the IR cut filter 61 and the performance of the lens 62. Good.

  FIG. 8 shows the experimental results of comparing the light shielding performance in the scanning microscope 1 by changing the installation conditions of the IR cut filter 61. More specifically, in FIG. 8, an elemental glass is installed instead of the sample 2, and the IR cut filter 61 a is used only by one sheet in a state where the IR laser light reflected by the elemental glass is incident on the NDD 17. In this case, when the two lenses 62 are stacked without sandwiching the lens 62 between the IR cut filter 61a and the IR cut filter 61b, or as described above, the lens 62 is sandwiched between the IR cut filter 61a and the IR cut filter 61b. The result of comparing the amount of light received by the PMT 66a is shown.

  As shown in FIG. 8, the amount of light received by the PMT 66a is the smallest when the lens 62 is sandwiched between the IR cut filter 61a and the IR cut filter 61b, and two IR cut filters 61a and IR cut filters 61b are stacked. The case is the next smallest, and the case where only one IR cut filter 61a is used is the largest. In other words, the light shielding performance against IR pulse light (noise light) is highest when the lens 62 is sandwiched between the IR cut filter 61a and the IR cut filter 61b, and when only one IR cut filter 61a is used. Is the lowest. In particular, when the lens 62 is sandwiched between the IR cut filter 61a and the IR cut filter 61b, it can be seen that the light shielding performance against IR pulse light (noise light) is dramatically improved.

  From the above results, it can be seen that not only two IR cut filters 61 are stacked, but also the light shielding performance against noise light can be easily improved by devising the installation conditions.

  In addition to sandwiching the lens 62 between the IR cut filter 61a and the IR cut filter 61b, for example, as shown in FIG. 9, the IR cut filter 61a and the IR cut filter 61b are connected to each other in front of the lens 62. The light shielding surface may be inclined. Thereby, the incident angle of the noise light to the IR cut filter 61a and the incident angle to the IR cut filter 61b are different, and the noise light transmitted through the IR cut filter 61a is more reliably shielded by the IR cut filter 61b. It becomes possible.

  In FIG. 9, the light shielding surface of the IR cut filter 61a is arranged so as to be perpendicular to the optical axis of the lens 62, but the light shielding surface of the IR cut filter 61b is not the lens but the IR cut filter 61a. You may make it arrange | position so that it may become perpendicular | vertical with respect to 62 optical axes. In addition, both the IR cut filter 61 a and the IR cut filter 61 b may be arranged to be inclined with respect to the optical axis of the lens 62.

  Further, the IR cut filter 61a and the IR cut filter 61b may be selected so that the performance with respect to the incident angle is different from each other. For example, the light-shielding bands that can shield incident light are substantially the same, and the light-shielding characteristics with respect to the incident angle are different from each other (for example, one has the highest light-shielding rate for light with an incident angle of 0 degrees, and the other has a predetermined oblique angle. The IR cut filter 61a and the IR cut filter 61b may be selected so that the light shielding rate with respect to the light having the incident angle in the direction is the highest. Thereby, the noise light transmitted through the IR cut filter 61a can be more reliably shielded by the IR cut filter 61b.

  Furthermore, the number of IR cut filters 61 is not limited to two, and three or more may be used.

  The lens 62 may be realized by a single lens or may be realized by a plurality of lens groups.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

  1 scanning microscope, 2 samples, 11 laser light source, 12 scanning device, 14 dichroic mirror, 15 objective lens, 17 NDD, 61a, 61b IR cut filter, 62 lens, 65a, 65b lens, 66a, 66b PMT

Claims (6)

In a scanning microscope that irradiates a sample while scanning excitation light capable of multiphoton excitation through a scanning unit, and detects the observation light emitted from the sample by the detection unit without scanning by the scanning unit,
On the optical path of the observation light to the detection means for detecting the observation light,
A first light shielding means having a property of shielding light in the same wavelength region as the excitation light and not having a property of selectively transmitting the observation light ;
A second light shielding means having a property of shielding light in the same wavelength range as the excitation light and not having a property of selectively transmitting the observation light ,
An incident angle of at least a part of the excitation light that passes through the first light shielding unit and enters the second light shielding unit is an incident angle to the first light shielding unit. Are differently configured scanning microscopes. The first light shielding means and the second light shielding means have the same performance,
The scanning microscope according to claim 1, further comprising a lens that collects light transmitted through the first blocking unit between the first blocking unit and the second blocking unit. The excitation light is condensed on the sample, the distance between the pupil of the objective lens through which the observation light emitted from the sample passes and the lens is d, the focal length of the lens is f, and the objective lens When the pupil diameter is φ1 and the effective diameter of the lens is φ2, the following formula is satisfied.

The scanning microscope according to claim 2. At least one light shielding surface of the light shielding surface of the first light shielding means and the light shielding surface of the second light shielding means is an optical axis of an optical system including the first light shielding means and the second light shielding means. The scanning microscope according to claim 1, wherein the scanning microscope is arranged to be inclined with respect to the scanning microscope. The scanning microscope according to claim 1, wherein the first light shielding unit and the second light shielding unit have substantially the same light shielding band and have different light shielding characteristics with respect to an incident angle. A barrier filter that selectively transmits the observation light is provided between the second light shielding unit and the detection unit.
The scanning microscope according to any one of claims 1 to 5.

Images